Recent developments in the laboratory of the present inventors have enhanced the ability of researchers to detect molecular events in solution and in real time without requiring molecular labels or extra process steps. The first developments involved a molecular binding layer used to capture potential ligands, with the molecular binding layer being electromagnetically coupled to a continuous transmission line that carried the appropriate electromagnetic signal. See, for example, U.S. application Ser. No. 09/243194, filed Feb. 2, 1999, and U.S. application Ser. No. 09/365578, filed Aug. 8, 1999. This development typically used a signal that did not penetrate deeply into the overlying solution, so that binding interactions could be easily detected regardless of the content of the overlying solution, which was essentially invisible under the experimental conditions (although other embodiments used a molecular binding layer separated from the transmission line). Other techniques directly detected molecular events in solution, using a signal that penetrates into the solution. See, for example, U.S. patent application Ser. No. 09/687,456, filed Oct. 13, 2000.
These new techniques make it possible to detect binding interactions without washing or other separation steps. In other words, it is possible to determine whether A and B, when mixed together, form an Axc2x7B complex or simply remain separate from each other but both in the same solution (here denoted A+B). This provides detection and observation of the actual binding event in solution in real time without labels, as opposed to prior art techniques, which are typically capture techniques that detect the result of binding after the event or techniques that require labeling of both components.
Many prior techniques have been able to determine whether A binds with B by capturing B onto a surface to which A is already attached (the surface in effect is a large label attached to A). Such techniques require a washing step prior to the detecting step, as the event actually being detected is the presence of B newly attached to the surface (through binding to A). This xe2x80x9cresults of bindingxe2x80x9d orientation of this technique is exemplified by so-called sandwich assays using antibodies and by hybridization of nucleic acids using a probe attached to a surface. Separating unattached B from A attached to the surface allowed the user to tell if B had become bound to A, simply by detecting the presence of B. However, such techniques detect the results of binding and are not detection of the binding event itself in real time, and the attachment of A to the surface can interfere with the ability of A to bind with B and other potential binding partners.
One capture technique that does detect binding in real time is surface plasmon resonance (SPR). This technique uses total internal reflection of light from a surface to which one potential binding partner is attached and detects changes in the critical angle of reflection when a capture event occurs on the surface. They thus are capture devices in requiring attachment of one component to a surface, although they are able to detect binding without wash steps.
Other techniques are available that detect binding in solution in real time, but these techniques typically require labeling of both potential binding partners or binding on a surface. For example, a fluorescent marker can be attached to A while a fluorescent quencher is attached to B. Quenching of fluorescence is an indication of binding. Such techniques, however, are disadvantageous in requiring labeling of one or both the A and B components, which is expensive and which may interfere with the binding event itself.
The newer techniques developed by the present inventors and others working together with them have not only made real-time, label-free binding detection possible (both in solution and on a surface), they also make it possible to provide information on the nature of the binding event. For example, it is possible to determine whether a given test compound binds to the active site on a particular drug receptor as an agonist or an antagonist or whether the test compound binds to an allosteric site, not simply just to indicate whether some uncharacterized binding event has taken place.
In addition to molecular interactions, another type of molecular event that these recent developments have enabled is the study of molecular structures. It is possible, by obtaining an electromagnetic signature of the molecule in the detection range using the now-enabled new techniques, to classify unknown molecules as having structure relationships with know class of molecules (e.g., to classify an unknown molecule as being a G-protein or as having particular features, such as a xcex2-sheet, in its structure).
However, although the promise of the technique has been high, the technology remains in its early stages, and improvements in methodology and equipment are continually needed. For example, early studies were often difficult to duplicate for reasons thatxe2x80x94because of the newness of the technologyxe2x80x94were not understood.
One of the factors that was considered by the inventors in an attempt to improve reproducibility was control of temperature of the sample, as it is known that the permittivity of a material, such as a test solution, changes with temperature. Such temperature control of the sample, however, did not appear to be sufficient to account for the difficulty in obtaining reproducible results.
It is known already to control temperature of the sample itself (or at least to monitor temperature and to use the temperature to correct experimental values) in an apparatus that determines the permittivity of polar solutions in order to determine the concentration of one polar material (e.g., ethanol) in a second polar material (e.g., water, as might be done in monitoring of a fermentation process). This is exemplified by U.S. Pat. No. 5,363,052 to McKee, entitled xe2x80x9cPermittivity Spectroscopy Apparatus and Method.xe2x80x9d This patent describes an apparatus and method for measuring the permittivity of a polar solution specimen to enable a determination of the concentration of polar constituents in the specimen. The apparatus employs a band pass filter including containment means formed to contain the polar solution and electrically dispose the polar solution as a dielectric element in the band pass filter; conducting means; a source of electrical current connected to said band pass filter; frequency variation means electrically connected to the electric voltage source to enable variation of the frequency at which current is applied to the band pass filter; and voltage sensing means electrically connected to sense the peak voltage passed by the band pass filter. The method includes providing a band pass filter having a conducting microstrip, disposing a specimen solution between the conducting microstrip and the ground plane; applying an electric current to the band pass filter; varying the frequency of the current; and determining the center frequency of the band pass filter as the current is varied.
In this patent, the permittivity of polar solution being investigated and thus the center frequency of the circuit in which the polar solution is an element are a function of temperature. Accordingly, the temperature of the polar solution in the microstrip circuit is controlled. A temperature regulator (temperature control means) of unspecified structure performs this task. A pump circulates polar solution between microstrip assembly and the temperature regulator in order to regulate the temperature of the polar solution at the measurement location.
McKee states that, in practice, maintaining the polar solution at a constant temperature is a difficult task, even with use of a temperature regulator (in fact, there is no description in the patent of the temperature range that the solution would be controlled to within, other than xe2x80x9croom temperaturexe2x80x9d or the xe2x80x9cfixed temperaturexe2x80x9d of a standard permittivity value obtained from a reference source; see, McKee, col. 6, line 8, and col. 7, lines 8-10). McKee therefore proposes an alternative method for compensating for temperature drift by calibrating his microstrip circuit to detect the temperature of polar solution and then calculating permittivity from a formula derived for the particular instrument being used. This is done by measuring the center frequency for the circuit over a range of temperatures and determining an equation from these measurements to relate center frequency to temperature for a given solution.
Even before discovering the McKee patent, applicant had attempted to control variability in measurements by controlling the temperature of the sample and had discovered that control of the sample temperature alone was not sufficient to provide a level of accuracy that allowed routine determination of the occurrence of molecular events. Detection of molecular events is an entirely different process from that of McKee and one that requires much greater accuracy of measurements, since the permittivity changes are much smaller than those obtained when determining the percent composition of mixtures of polar solvents. Specifically, it was found that control of the temperature of a solution being measured via a constant temperature bath, with the solution being transported from the bath location to a second location in which a measurement of electrical parameters is actually being made, did not solve the problem of variable results when attempting to detect molecular events.
Accordingly, there exists a need for further development of the methods of detecting molecular events using the basic technology that originated in the laboratories of the present inventors. The present invention fulfills many of the needs discussed above and others as well, as described herein.
Surprisingly, it has been discovered that inability to make readily repeatable experimental conclusions, when taking precise electrical parameter measurements for the purpose of detecting molecular events, can be substantially overcome by appropriately controlling temperature of both the sample being measured and the signal transmitting and receiving portions of the electronic circuitry of the detection apparatus, even when control of the temperature of the sample is insufficient. The temperature control must be precise and comprehensive in order to achieve reproducibility. Since a change in electrical measurement parameters can result from the molecular event or from a temperature change, it is difficult if not impossible to determine which event is causing the detected change in the absence of appropriate temperature controls. Correction for temperature via calculation is difficult under many measurement conditions, since different parts of the measurement probe and sample can be at different temperatures in an uncontrolled environment, meaning that calculated temperature corrections based on sample readings do not account for enough error, due to the precision of measurement required when molecular events are being detected. Additionally, one must recall that a temperature probe actually measures its own temperature, and a temperature gradient may exsit between the probe and the material with which it is in contact.
Accordingly, the present invention provides a method for detecting a molecular event, comprising (1) applying an electromagnetic test signal in a frequency range from 1 MHz to 1000 GHz to a sample in which a molecular event is being detected, whereby the sample interacts with and modulates the test signal to produce a modulated test signal, and (2) detecting the modulated test signal, wherein the applying and detecting take place in a temperature-controlled environment, wherein the temperature-controlled environment comprises the sample, a radiating portion of a signal generating circuit, and a receiving portion of a signal detection circuit and wherein the applying and detecting take place in the environment at a temperature controlled to within xc2x10.5xc2x0 C.